Final Report, Identification of Specimen; Environmental Isolate Biology 309, Microbiology, Fall 2004 Biff Studmuffin, Section 024

3 December 2004 Introduction: Given that the normal micro flora of the human skin is comprised of over 100 separate genera and several times that number of species, a bacterial specimen was obtained by scraping the surface of the skin on the forearm of a volunteer. This sample was incubated on Nutrient Agar (NA) at 37 degrees Centigrade for 48 hours. After incubation the plate was examined and a highly pigmented colony was subsequently isolated by streak plate method. This specimen was maintained on NA slants throughout the semester testing period. Analytical testing included both colony and cellular morphology as well as metabolic and environmental growth determinations. Consultation with Bergeys Manual of Systematic Bacteriology and with other listed sources lead to the conclusion that this specimen is most likely Staphylococcus aureus. S. aureus is a normal inhabitant of the human epidermis and as such does not normally produce pathology. However it is an opportunistic pathogen of humans, typically colonizing wounds or other breaks in the skin. Such colonization typically results in encapsulated or abscess type injury. However, this bacteria produces a variety of strain dependent exotoxins and has been implicated in both Scalded Skin Syndrome specifically and toxic shock syndromes in general. S. aureus is one of the most common nosocomial pathogens associated with surgical wounds and has developed resistance to the majority of standard antibiotics. Some strains resistant to Vancomyacin have been reported as well. A common and normal member of skin micro flora, it appears to just be waiting for the opportunity cause disease. Methods: Protocols are not listed but were modified and applied according to the Bio 309 lab manual (see references). Cultural growth characteristics were determined by subculture on Nutrient Agar and Nutrient Agar slant. Cellular growth characteristics were determined after the specimen was stain by negative and simple staining. Gram staining characteristics were determined by standard Gram staining which confirmed cellular character. Endospore formation was determined using the endospore staining procedure outlined in the reference. Acid Fast staining was performed but found not to be diagnostic for this specimen. Aerobic character of this specimen was analyzed by inoculation of a molten NA deep and was confirmed during motility testing utilizing a motility agar deep. Differential media included Mannitol Salt, Eosin Methylene Blue, and MacConkeys Agar plates. Optimal growth temperature was analyzed by incubation of the specimen at 4 C, 24 C,

37 C, and 60 C. Optimum pH for growth was determined by inoculation of Nutrient Broth tubes adjusted to ph = 3, 5, 6, 7, and 9 and subsequent incubation at optimum growth temperature. Optimum osmotic growth and salt tolerance was analyzed after inoculation of NA plates adjusted to 0.0825%, 5%, 10%, and 15% NaCl concentration. Metabolic Analysis consisted of both substrate utilization and enzymatic capabilities of and by the specimen. Hemolysis capability was examined subsequent to inoculation and incubation on a Tryptic Soy Agar plate containing 5% sheeps blood. Carbohydrate utilization as well as acid and sulfide production were tested using carbohydrate fermentation tubes with Durham tube collection as well as inoculation of Triple Sugar Iron (TSI) tubes. Nutrient requirements were determined for this specimen by inoculation of starch, urea, Milk agar, and Gelatin agar plates followed by incubation at the optimal growth temperature. Casein utilization was verified by the use of Litmus Milk tubes. Sulfide production was tested using both the TSI and Motility Agar tubes. The IMViC series of tests (Indole, Methyl Red, Voges-Proskauer, and Citrate) were performed on this specimen as per the reference. Finally enzymatic presence was tested by standard Catalase and Oxidase tests as described in the reference. Antibiotic sensitivity testing included incubation in the presence of PML antibiotic disks containing Pennicillin, Methicillin, Bacitracin, Sulfaoxazole, Gentamycin, Vancomycin, and Novobiocin. Results: Cultural characteristics on NA plates and NA slants consistently consisted of smooth, convex, round, and sometimes glistening colonies always highly pigmented orange or gold. Negative and Simple (Methylene Blue) staining revealed this specimen as a bacillus shaped bacteria, sometimes growing in chains. Subsequent Gram staining indicated that this bacterium is Gram Positive. Acid Fast staining proved negative. However Endospore staining showed the presence of a high number of spores. Note that this sample of the specimen was taken from an NA plate stored at 4 C for more than three weeks. The specimen was found to grow well on Mannitol Salt Agar and utilized Mannitol with acid production. The specimen did not display any significant growth on EMB or MacConkeys Agar. Inoculation of an NA Deep indicated the presence of strictly aerobic bacteria. Physical factor analysis indicated that this bacterium is highly salt tolerant (15% NaCl), grows best at neutral to slightly acidic pH and at 37 C. The specimen was found to restrict its growth primarily to the stab entry point of the motility agar deep and was so classified as non-motile. These bacteria displayed the ability to utilize all three sugars in the TSI tube but did not produce any significant amounts of sulfides or gas. The carbohydrate fermentation tubes confirmed the specimens ability to utilize glucose, lactose, and sucrose as indicated by the TSI tube. No gas was produced in any fermentation tube. The Milk Agar plate was suggestive of Casein hydrolysis but this was contradicted by the overall negative reaction with the Litmus Milk tube.

Gelatinase and Amylase tests proved negative. The specimen displayed beta hemolysis on TSA with 5% sheeps blood. The presence of urease was indicated by a positive Urea slant. IMViC series characterized the specimen as Indole negative, MR positive, VP positive, and Citrate negative. Catalase and Oxidase test both proved positive for both enzymes. Of the antibiotics tested, this specimen proved sensitive only to Gentamycin and Novobiocin. While resistance to both Vancomycin and Methicillin are noted, this is likely due to the improper storage and age of these antibiotics. Conclusions: The specimen represents a sample of Staphylococcus aureus. The battery of staining procedures places this specimen in Section 12 of Bergeys Manual of Systematic Bacteriology. Of the 3 families listed, growth in clusters and acid from carbohydrate fermentation places the specimen within the family Micrococcus. Of the four genera listed for Micrococcus, growth in random clusters, fermentation of glucose, and motility place the specimen within the genus staphylococcus. While testing was not as extensive as desirable, speciation is possible. The most significant factor in speciation was the high degree of pigmentation displayed during subculture of the specimen. The orange to gold pigmentation of the colonies on NA alone is highly suggestive of S. aureus, isolating it from all but one of the 19 listed species. The production of Acetoin is similarly limited to only five of the 19 species. Finally, only S. aureus consistently displays hemolysis on the blood agar plate. Definitive testing to produce a high degree of species certainty would consist of Coagulase testing. References: Test Protocols Microbiology; A Laboratory Manual, Cappuccino and Sherman, 7th Edition. Speciation Bergeys Manual of Systematic Bacteriology, 1st Edition, Williams and Wilkins, Baltimore, 1986 Antibiotic Resistance http://www.cdc.gov/ncidod/hip/ARESIST/visa.htm

Format Requirements Final Report of Speciation of Environmental Isolate 1. Introduction ( 20 points) a. Source of sample 2 points b. Description of collection and maintenance of specimen 5 points c. Identification of specimen 3 points d. Background information Pathogen potential 10 points 2. Methods (10 points) a. Description of tests performed and purpose of each 5 points b. Either detailed protocol or properly cited reference 5 points 3. Results ( 20 points) a. Complete listing of results for every test 10 points b. Confidence level of each test 5 points c. Inclusion / explanation of conflicting test results 5 points 4. Conclusions (40 points) a. Most extensive identification possible 10 points b. Use of data to justify conclusion 25 points c. Compliance with Bergeys 5 points 5. References (10 points) a. Protocol reference if no detailed protocol described 5 points b. All declarative statements properly cited 5 points